Combining hypoxic methods for peak performance.

New methods and devices for pursuing performance enhancement through altitude training were developed in Scandinavia and the USA in the early 1990s. At present, several forms of hypoxic training and/or altitude exposure exist: traditional 'live high-train high' (LHTH), contemporary 'live high-train low' (LHTL), intermittent hypoxic exposure during rest (IHE) and intermittent hypoxic exposure during continuous session (IHT). Although substantial differences exist between these methods of hypoxic training and/or exposure, all have the same goal: to induce an improvement in athletic performance at sea level. They are also used for preparation for competition at altitude and/or for the acclimatization of mountaineers. The underlying mechanisms behind the effects of hypoxic training are widely debated. Although the popular view is that altitude training may lead to an increase in haematological capacity, this may not be the main, or the only, factor involved in the improvement of performance. Other central (such as ventilatory, haemodynamic or neural adaptation) or peripheral (such as muscle buffering capacity or economy) factors play an important role. LHTL was shown to be an efficient method. The optimal altitude for living high has been defined as being 2200-2500 m to provide an optimal erythropoietic effect and up to 3100 m for non-haematological parameters. The optimal duration at altitude appears to be 4 weeks for inducing accelerated erythropoiesis whereas <3 weeks (i.e. 18 days) are long enough for beneficial changes in economy, muscle buffering capacity, the hypoxic ventilatory response or Na(+)/K(+)-ATPase activity. One critical point is the daily dose of altitude. A natural altitude of 2500 m for 20-22 h/day (in fact, travelling down to the valley only for training) appears sufficient to increase erythropoiesis and improve sea-level performance. 'Longer is better' as regards haematological changes since additional benefits have been shown as hypoxic exposure increases beyond 16 h/day. The minimum daily dose for stimulating erythropoiesis seems to be 12 h/day. For non-haematological changes, the implementation of a much shorter duration of exposure seems possible. Athletes could take advantage of IHT, which seems more beneficial than IHE in performance enhancement. The intensity of hypoxic exercise might play a role on adaptations at the molecular level in skeletal muscle tissue. There is clear evidence that intense exercise at high altitude stimulates to a greater extent muscle adaptations for both aerobic and anaerobic exercises and limits the decrease in power. So although IHT induces no increase in VO(2max) due to the low 'altitude dose', improvement in athletic performance is likely to happen with high-intensity exercise (i.e. above the ventilatory threshold) due to an increase in mitochondrial efficiency and pH/lactate regulation. We propose a new combination of hypoxic method (which we suggest naming Living High-Training Low and High, interspersed; LHTLHi) combining LHTL (five nights at 3000 m and two nights at sea level) with training at sea level except for a few (2.3 per week) IHT sessions of supra-threshold training. This review also provides a rationale on how to combine the different hypoxic methods and suggests advances in both their implementation and their periodization during the yearly training programme of athletes competing in endurance, glycolytic or intermittent sports.

Paradoxical effects of endurance training and chronic hypoxia on myofibrillar ATPase activity.

This study aimed to determine the changes in soleus myofibrillar ATPase (m-ATPase) activity and myosin heavy chain (MHC) isoform expression after endurance training and/or chronic hypoxic exposure. Dark Agouti rats were randomly divided into four groups: control, normoxic sedentary (N; n = 14), normoxic endurance trained (NT; n = 14), hypoxic sedentary (H; n = 10), and hypoxic endurance trained (HT; n = 14). Rats lived and trained in normoxia at 760 mmHg (N and NT) or hypobaric hypoxia at 550 mmHg (approximately 2,800 m) (H and HT). m-ATPase activity was measured by rapid flow quench technique; myosin subunits were analyzed with mono- and two-dimensional gel electrophoresis. Endurance training significantly increased m-ATPase (P < 0.01), although an increase in MHC-I content occurred (P < 0.01). In spite of slow-to-fast transitions in MHC isoform distribution in chronic hypoxia (P < 0.05) no increase in m-ATPase was observed. The rate constants of m-ATPase were 0.0350 +/- 0.0023 s(-1) and 0.047 +/- 0.0050 s(-1) for N and NT and 0.033 +/- 0.0021 s(-1) and 0.038 +/- 0.0032 s(-1) for H and HT. Thus, dissociation between variations in m-ATPase and changes in MHC isoform expression was observed. Changes in fraction of active myosin heads, in myosin light chain isoform (MLC) distribution or in MLC phosphorylation, could not explain the variations in m-ATPase. Myosin posttranslational modifications or changes in other myofibrillar proteins may therefore be responsible for the observed variations in m-ATPase activity.

Photoperiod-dependent changes in melatonin synthesis in the turkey pineal gland and retina.

The effect of photoperiod on melatonin content and the activity of the melatonin-synthesizing enzymes, namely, serotonin N-acetyltransferase (AANAT) and hydroxyindole-O-methyltransferase, were investigated in the pineal gland and retina of turkeys. The birds were adapted to 3 different lighting conditions: 16L:8D (long photoperiod), 12L:12D (regular photoperiod), and 8L:16D (short photoperiod). Pineal, retinal, and plasma melatonin concentrations oscillated with a robust diurnal rhythm, with high values during darkness. The duration of elevated nocturnal melatonin levels in the turkey pineal gland, retina, and plasma changed markedly in response to the length of the dark phase, being longest during the short photoperiod with 16 h of darkness. These photoperiodic variations in melatonin synthesis appear to be driven by AANAT, because changes in the activity of this enzyme were closely correlated with changes in melatonin. By contrast, pineal and retinal hydroxyindole-O-methyltransferase activities failed to exhibit any significant 24-h variation in the different photoperiods. A marked effect of photoperiod on the level of melatonin production was also observed. Peak values of melatonin and AANAT activity in the pineal gland (but not in the retina) were highest during the long photoperiod. During the light phase, mean melatonin concentrations in the pineal gland and retina of turkeys kept under the long photoperiod were significantly higher compared with those from birds maintained under the regular and short photoperiods. In addition, mean circulating melatonin levels were lowest in the short photoperiod. Finally, the magnitude of the light-evoked suppression of nighttime pineal AANAT activity was also influenced by photoperiod, with suppression being smallest under the long photoperiod. These findings show that in the turkey, photoperiod plays an important role in regulating the melatonin signal.

Heart rate variability and performance at two different altitudes in well-trained swimmers.

UNLABELLED: The aim of this study was to compare the effects of training at two different altitudes on heart rate variability (HRV) and performance in well-trained swimmers. Eight national-level male swimmers (age = 17.0 +/- 1.8 yrs, weight = 67.0 +/- 6.6 kg, height = 180.4 +/- 7.2 cm, V(O2max) = 60.4 +/- 4.0 ml.min(-1). kg(-1)) trained 17 days at 1200 m altitude (T1200), then, after 6 weeks of moderate training at sea level, reproduced the same training plan at 1850 m (T1850). The training was mainly aerobic with 86 % and 84 % < or = anaerobic threshold for T 1200 and T1850, respectively. Four HRV analysis tests were performed during T1200 and T1850, respectively (pre-test = day 0, test 2 = day 5, test 3 = day 11, post-test = day 17), in supine and standing position. Performance was measured over a 2000-m freestyle test at the altitude of 1200 m. A difference in HRV changes was observed between the two altitudes: during T1200, addition of parasympathetic and sympathetic activity in supine (TP(SU)) (p < 0.05) and standing (TP(ST)) (p < 0.05) position, supine parasympathetic activity (HF(SU)) (p < 0.05), and standing sympathetic activity (LF(ST)) (p < 0.05) were increased and the 2000-m performance was improved (p < 0.05) whereas none of these parameters was changed during T1850. Change in performance was correlated with increase in HF(SU) (r = 0.73; p < 0.05) and tended towards correlation with increase in LF(ST) (r = 0.73; p = 0.06). CONCLUSION: the same training loads induced a positive effect on HRV and performance at 1200 m but not at 1850 m. This may be the consequence of greater stress due to an interaction between greater hypoxic stimulus and the same training loads. These results highlight two opposing effects: aerobic training increases, whereas hypoxia decreases HF(SU), due to the correlation between HRV and changes in performance during altitude training.

Is it more effective for highly trained swimmers to live and train at 1200 m than at 1850 m in terms of performance and haematological benefits?

OBJECTIVES: The effects of living and training have not been compared at different altitudes in well trained subjects. METHODS: Nine international swimmers lived and trained for 13 days similarly at 1200 m (T1200) and 1850 m (T1850). The two altitude training periods were separated by six weeks of sea level training. Before and after each training trip, subjects performed, at an altitude of 1200 m, an incremental exercise test to exhaustion of 5 x 200 m swims and a maximal test over 2000 m. RESULTS: There was no difference in Vo(2)max after each training trip: the before values were 58.5 (5.6) and 60.4 (6.7) ml/kg/min and the after values were 56.2 (5.2) and 57.1 (4.7) ml/kg/min for T1200 and T1850 respectively. The 2000 m performance had improved during T1200 (1476 (34) to 1448 (45) seconds) but not during T1850 (1458 (35) v 1450 (33) seconds). Mean cell volume increased during T1850 (86.6 (2.8) to 88.7 (2.9) microm(3)) but did not change during T1200 (85.6 (2.9) v 85.7 (2.9) microm(3)). The proportion of reticulocytes decreased during T1200 (15.2 (3.8)% to 10.3 (3.4)%) and increased during T1850 (9.3 (1.6)% to 11.9 (3.5)%). CONCLUSIONS: The short term effects of 13 days of training at 1200 m on swimming performance appear to be greater than the same type of training for the same length of time at 1850 m. As mean cell volume and proportion of reticulocytes only increased during training at 1850 m, the benefits of training at this altitude may be delayed and appear later on.

Specificity of VO2MAX and the ventilatory threshold in free swimming and cycle ergometry: comparison between triathletes and swimmers.

OBJECTIVES: To compare maximal heart rate (HRmax), maximal oxygen consumption (VO2MAX), and the ventilatory threshold (VT; %VO2MAX) during cycle ergometry and free swimming between swimmers and triathletes. METHODS: Nine swimmers and ten triathletes completed an incremental swimming and cycling test to exhaustion. Whole body metabolic responses were determined in each test. RESULTS: The swimmers exhibited a significantly higher VO2MAX in swimming than in cycling (58.4 (5.6) v 51.3 (5.1) ml/kg/min), whereas the opposite was found in the triathletes (53.0 (6.7) v 68.2 (6.8) ml/kg/min). HRmax was significantly different in the maximal cycling and swimming tests for the triathletes (188.6 (7.5) v 174.8 (9.0) beats/min). In the maximal swimming test, HRmax was significantly higher in the swimmers than in the triathletes (174.8 (9.0) v 184.6 (9.7) beats/min). No significant differences were found for VT measured in swimming and cycling in the triathletes and swimmers. CONCLUSION: This study confirms that the exercise testing mode affects the VO2MAX value, and that swimmers have very specific training adaptations even compared with triathletes. This may be a function of acute physiological responses combined with the specialist training status of the different athletes influencing maximal cardiac output or oxygen extraction. In contrast, the different training regimens do not seem to influence the VT, as this variable did not differ between the two testing modes in either group.

Seasonal variations in the nycthemeral rhythm of plasma melatonin in the camel (Camelus dromedarius).

Seasonal changes in the pattern of plasma melatonin were investigated in two groups of camels (Camelus dromedarius): 11 adult and six young camels. Animals were subjected to the outdoor conditions of a desert environment. Blood samples were taken at regular intervals of about 3 hr (added to particular samples at 1 hr before then 30 min and 1 hr after sunset, and 1 hr before and 1 hr after sunrise) for 24 hr at both solstices and equinoxes of the year. The plasma melatonin levels steeply increased soon after sunset and remained elevated throughout all the night. Then, melatonin concentrations progressively declined shortly before sunrise and returned to daytime basal levels 1 hr later. There was no seasonal variation in the amplitude or in the offset of the melatonin peak or in the daytime basal levels. The onset of the nocturnal peak was delayed by 2 hr in June at the summer solstice (P < 0.05), which can be related to the changes in night length between the two solstices. A significant effect of age was observed in all seasons. Melatonin levels were higher in the young camel group (fall equinox: P < 0.001; spring equinox: P < 0.01; winter solstice: P < 0.01; summer solstice: P < 0.05). The pattern of melatonin secretion in the camel showed a significant seasonal variation parallel to the photoperiodic changes of the year. The observed decline of melatonin levels during an extra-light pulse in the middle of the night indicates the light control of melatonin synthesis. It is not yet known if, in this low latitude desert region, the seasonal breeding period of the camel is cued by the photoperiod. The data obtained, however, clearly demonstrate that the camel has the capacity to follow and to integrate photoperiodic changes through melatonin changes.

Physiological responses during submaximal interval swimming training: effects of interval duration.

The aim of the present study was to determine the time sustained near VO2max in two interval training (IT) swimming sessions comprising 4x400 m (IT(4x400)) or 16x100 (IT(16xl00)). Elite swimmers (Mean+/-SD age 18+/-2 yrs; body mass 66.9+/-6.5 kg: swim VO2max 55.7+/-5.8 ml.kg(-1).min(-1)) completed three experimental sessions at a 50-m indoor pool over a one week period. The first test comprised a 5 x 200-m incremental test to exhaustion for determination of the pulmonary ventilation threshold (VT, m.s(-1)), VO2max, the velocity associated with VO2max (VO2max, m(s(-1)) and maximum heart rate (HR(max), b.min(-1)). The remaining two tests involved the IT(4x400) and IT(16xl00) performed in a randomised order. The two IT sessions where completed at a velocity representing 25% of the difference between the VT and the VO2max (delta25%) and in the same work to rest ratio. During the IT sessions VO2 as well as HR were measured. The duration (s) >90% VO2max, also the duration (s) >90% HR(max), were not significantly different in the IT(16x100) and IT(4x400). However, limits of agreement (LIM(AG)) analysis demonstrated considerable individual variation in the time >90% VO2max (mean difference +/-2SD = 222+/-819 s) and the time >90% HRmax (mean difference +/-2SD = 61+/-758 s) between the two IT sessions. This factor deserves further research to establish the characteristics of those athletes which influence the physiological responses in IT of short or longer duration repetitions.

Melatonin and 5-methoxytryptophol (5-ML) in nervous and/or neurosensory structures of a gastropod mollusc (Helix aspersa maxima): synthesis and diurnal rhythms.

Daily patterns of melatonin and 5-methoxytryptophol (5-ML) concentrations and of aryl alkylamine N-acetyltransferase (AA-NAT) and hydroxyindole-O-methyltransferase (HIOMT) activities have been measured in the cerebroid ganglions, visceral ganglions, and ocular tentacles of the gastropod mollusc Helix aspersa maxima. Melatonin concentrations are very low in all the studied structures, except a small peak at the end of the night in the cerebroid ganglions. 5-ML, which is quite undetectable in the cerebroid and visceral ganglions, shows clear daily variations in the ocular tentacles with low values in the middle of the light period and high values during the night. These results are opposite to what is known on daily variations of 5-ML in vertebrates. AA-NAT activity was not detected, while the presence of an HIOMT-like activity supports the hypothesis that 5-ML is synthesized in the ocular tentacles. The temporal relationships existing between the 5-ML rhythm in the ocular tentacles and the hemolymph suggest that 5-ML could be released in the general circulation. These preliminary results suggest that 5-ML could be an informative molecule involved in adaptative processes in the snail and they reinforce the hypothesis that the different 5-methoxyindoles could be implicated in the integration of environmental information.

Effect of melatonin supplementation on the sexual development in European quail (Coturnix coturnix).

At the end of their wintering phase, male European quails were exposed to a stimulation photoperiod of light/dark 12:12 h for 10 days to induce sexual development. A daily oral melatonin supplementation was then given to one group of treated males (N=11) and the alcohol solvent was given to a control group of males (N=10). These solutions were provided during the final 3 h of the photophase for 28 days, then during the final 4 h for 18 days. There were no significant differences between the two groups with respect to fat levels. However, 3 weeks after the beginning of melatonin supplementation, the sexual development of the treated birds slowed down. The importance of this decline varied to a greater or lesser degree between individual birds. When melatonin supplementation stopped, sexual development resumed. Activity recordings revealed a decrease in feeding activity when melatonin supplementation was provided. However, this response showed important interindividual variability. The birds that produced the most marked responses to melatonin during the first 3 weeks of supplementation were those that also showed the most obvious decline in sexual development. It seems that, in European quail, a wild migratory species that always shows a natural biological annual rhythm, a melatonin signal could play a role in regulating reproduction.

Effects of cycloheximide and aminophylline on 5-methoxytryptophol and melatonin contents in the chick pineal gland.

The chick pineal gland rhythmically synthesizes two 5-methoxyindoles, melatonin and 5-methoxytryptophol. These rhythms are circadian in nature and have opposite phases. The aim of this study was to determine the effects of cycloheximide, a protein synthesis inhibitor, and aminophylline, an inhibitor of phosphodiesterase, on 5-methoxytryptophol content in the chick pineal gland and to compare this with the drugs' action on pineal melatonin production. Inhibition of melatonin biosynthesis by cycloheximide (1 mg/kg, i.p. ), revealed by a marked reduction in the nighttime activity of serotonin N-acetyltransferase (AA-NAT; a key regulatory enzyme in melatonin synthesis) and melatonin concentrations, was accompanied by a significant increase in 5-methoxytryptophol content. In contrast, administration of aminophylline (100 mg/kg, i.p.) to light-exposed chicks significantly increased pineal AA-NAT activity and melatonin levels and decreased 5-methoxytryptophol concentrations. It is concluded that in the chick the production of pineal 5-methoxytryptophol and melatonin is inversely correlated.

Long-term daily melatonin infusion induces a large increase in N-acetyltransferase activity, hydroxyindole-O-methyltransferase activity, and melatonin content in the Harderian gland and eye of pinealectomized male Siberian hamsters (Phodopus sungorus).

The effects of long-term daily melatonin infusions on the melatonin synthetic pathway in the Harderian glands and eyes of male Siberian hamsters were studied. Hamsters were pinealectomized (PX) and infused daily for 8 hr with either melatonin (6 microg/hr) or vehicle for 7 days in short photoperiod (SP, 10L:14D), followed by 14 wk in either SP (SP group) or in constant darkness (DD group). After the infusion period (15 wk), the infusion was stopped and animals were transferred into SP for 3 wk. The hamsters were then killed at midday or midnight. Exogenous melatonin infusion caused an increase in the Harderian gland weight, which was still evident 3 wk after the end of the treatment. In addition, exogenous melatonin increased endogenous melatonin concentrations (4-fold) and hydroxyindole-O-methyltransferase (HIOMT) activity (2-fold). N-acetyltransferase (NAT) activity, however, was not increased, and no day/night difference in melatonin content and HIOMT activity was observed in the Harderian glands. In the eye, melatonin infusions significantly increased day and night-time melatonin levels (up to 3-fold) and both NAT and HIOMT activities (up to 3.5-fold). This effect of melatonin treatment was observed in both SP and DD groups. These observations demonstrate that exogenously-infused melatonin at relatively high doses activates the synthesis of endogenous melatonin in the Harderian gland and eye of the Siberian hamster. Circulating levels of melatonin were also markedly increased, indicating that in these conditions melatonin may be released from extra-pineal sites.

Phase-shifting effects of light on the circadian rhythms of 5-methoxytryptophol and melatonin in the chick pineal gland.

In the chick pineal gland, 5-methoxytryptophol and melatonin concentrations fluctuate in a rhythmic manner. These rhythms are circadian in nature persisting in constant darkness and have opposite phases. Acute exposure of chicks to white light (30 lux for 5, 10, 20, and 30 min) at night increased the amount of pineal 5-methoxytryptophol and decreased pineal melatonin content. A 6 hr pulse of light (100 lux) applied early in the subjective night (CT12-CT18) caused a delay in the phase of the circadian rhythms of 5-methoxytryptophol and melatonin by 3.7 and 4.5 h, respectively, compared to untreated controls. When the 6 hr light pulse was given during the late subjective night (C18 CT24) it advanced the phase of the 5-methoxytryptophol and melatonin rhythms by 8.1 and 11.9 h, respectively. In the chick pineal the phase-advancing effects of light on the circadian rhythms of 5-methoxytryptophol and melatonin were more pronounced than the phase-delaying effects. Our results provide the first evidence that light is capable of phase shifting the 5-methoxytryptophol rhythm in a manner similar to its action on the melatonin rhythm.

Transcription factor dynamics and neuroendocrine signalling in the mouse pineal gland: a comparative analysis of melatonin-deficient C57BL mice and melatonin-proficient C3H mice.

In rodents, the nocturnal rise and fall of arylalkylamine N-acetyltransferase (AANAT) activity controls the rhythmic synthesis of melatonin, the hormone of the pineal gland. This rhythm involves the transcriptional regulation of the AANAT by two norepinephrine (NE)-inducible transcription factors, e.g. the activator pCREB (phosphorylated Ca2+/cAMP-response element binding protein) and the inhibitor ICER (inducible cAMP early repressor). Most inbred mouse strains do not produce melatonin under standard laboratory light/dark conditions. As melatonin-deficient mice are often the founders for transgenic animals used for chronobiological experimentations, molecular components of neuroendocrine signalling in the pineal gland as an integral part of clock entrainment mechanisms have to be deciphered. We therefore compared calcium signalling, transcriptional events and melatonin synthesis in the melatonin-deficient C57BL mouse and the melatonin-proficient C3H mouse. Pineal glands and primary pinealocytes were cultured and stimulated with NE or were collected at various times of the light/dark (LD) cycle. Changes in intracellular calcium concentrations, the phosphorylation of CREB, and ICER protein levels follow similar dynamics in the pineal glands of both mouse strains. pCREB levels are high during the early night and ICER protein shows elevated levels during the late night. In the C57BL pineal gland, a low but significant increase in melatonin synthesis could be observed upon NE stimulation, and, notably, also when animals were exposed to long nights. We conclude that the commonly used C57BL mouse is not completely melatonin-deficient and that this melatonin-deficiency does not affect molecular details involved in regulating transcriptional events of melatonin synthesis.

Overexpression of neuropeptide Y induced by brain-derived neurotrophic factor in the rat hippocampus is long lasting.

Brain-derived neurotrophic factor (BDNF) plays an important role in hippocampal neuroplasticity. In particular, BDNF upregulation in the hippocampus by epileptic seizures suggests its involvement in the neuronal rearrangements accompanying epileptogenesis. We have shown previously that chronic infusion of BDNF in the hippocampus induces a long-term delay in hippocampal kindling progression. Although BDNF has been shown to enhance the excitability of this structure upon acute application, long-term transcriptional regulations leading to increased inhibition within the hippocampus may account for its suppressive effects on epileptogenesis. Therefore, the long-term consequences of a 7-day chronic intrahippocampal infusion of BDNF (12 microg/day) were investigated up to 2 weeks after the end of the infusion, on the expression of neurotransmitters contained in inhibitory hippocampal interneurons and which display anti-epileptic properties. Our results show that BDNF does not modify levels of immunostaining for glutamic acid decarboxylase, the rate-limiting enzyme for gamma-aminobutyric acid (GABA) synthesis, and somatostatin. Conversely, BDNF induces a long-lasting increase of neuropeptide Y (NPY) in the hippocampus, measured by immunohistochemistry and radioimmunoassay, outlasting the end of the infusion by at least 7 days. The distribution of BDNF-induced neuropeptide Y immunoreactivity is similar to the pattern observed in animals submitted to hippocampal kindling, with the exception of mossy fibres which only become immunoreactive following seizure activity. The enduring increase of neuropeptide Y expression induced by BDNF in the hippocampus suggests that this neurotrophin can trigger long-term genomic effects, which may contribute to the neuroplasticity of this structure, in particular during epileptogenesis.

Potentiation effect of vasopressin on melatonin secretion as determined by trans-pineal microdialysis in the Rat.

The mammalian pineal gland is known to receive a noradrenergic innervation originating from the superior cervical ganglion which corresponds to the primary regulatory input for melatonin synthesis. However, many peptidergic fibers containing peptides such as vasopressin and oxytocin have also been found in the rat pineal gland. The present study was performed to investigate the possible role of vasopressin and oxytocin on melatonin secretion in vivo. Therefore, both neuropeptides were delivered for 2 h through a trans-pineal microdialysis probe directly into the gland at different times during the nocturnal phase of the light:dark cycle. At the same time pineal dialysates were collected continuously. Melatonin concentrations were measured by radioimmunoassay. Melatonin synthesis potentiation was achieved when vasopressin was infused locally in the pineal, during the onset of nocturnal melatonin secretion. In order to assess the possible role of a physiological increase of endogenous circulating vasopressin on pineal metabolism, melatonin synthesis was recorded in the same animals before and after a prolonged dehydration period. Night time melatonin concentration was increased after the water deprivation vs control conditions. Contrary to that, oxytocin seems not to affect pineal metabolism in the rat since no significant change was observed on melatonin secretion in response to a local oxytocin infusion. These results show that vasopressin can modulate melatonin synthesis in the rat pineal whereas no effect was obtained with oxytocin, at least under the present experimental conditions.

Interindividual differences in the pattern of melatonin secretion of the Wistar rat.

In vivo trans-pineal microdialysis was performed in male Wistar rats maintained under a 12 hr light:12 hr dark (LD 12:12) cycle. Collected dialysates were assayed by radioimmunoassay for melatonin concentrations. A non-linear regression was fitted through the obtained datapoints to determine the time points at which a 50% increase (IT50) and decrease (DT50) of the nocturnal melatonin peak were reached. In a first experiment, the nocturnal melatonin profiles of four animals were determined throughout 5 consecutive days. In a second experiment, we analysed the melatonin profiles during the night in rats originating from three different breeding colonies (Dépré Harlan, and Iffa-Credo). A low intraindividual variability was found on the phase markers IT50 and DT50, as on peak duration of melatonin rhythms estimated over 5 subsequent days in the same animal. In contrast, animals showed a large interindividual variability in their profile phase markers and the values were dependent on the origin of the breeding colony. Each rat colony was characterized by early or late IT50 and DT50 as long or short peak length. It is concluded from experiment 1 that the melatonin rhythm is a very stable circadian marker. Nevertheless, great caution must be taken in the choice of animal groups while studying circadian rhythms due to the large interindividual variability observed in experiment 2. Therefore, as the technique allows the use of the animal as its own control, the present study demonstrated that the use of the microdialysis technique is of interest in studies on the circadian system.

Daily and light-at-night induced variations of circulating 5-methoxytryptophol (5-ML) in ewes with respectively high and low nocturnal melatonin secretion.

The aim of the present study was to determine whether the genetic differences previously reported in ewe plasma melatonin concentrations were correlated with differences in the synthesis and release of other 5-methoxyindoles. To determine if 5-methoxytryptophol (5-ML), which is known to be present in large amounts in the sheep pineal gland, is released, as is melatonin, into the general circulation, and if some temporal relationships between 5-ML and melatonin release could be observed, two groups of ewes were selected with respect to their endogenous melatonin secretion: in the first experiment, ten ewes from the low melatonin group (low group) and ten ewes from the high melatonin group (high group). 5-ML was measured every hour during a 24-hr period by radioimmunoassay. In all ewes, 5-ML was released during day-time, the rhythm of 5-ML concentrations being inversely related with the melatonin rhythm. Both day-time and night-time 5-ML concentrations were higher in the ewes from the high group than in the ewes from the low group (14.7 +/- 1.0 pg/mL plasma versus 6.4 +/- 0.3 pg/mL plasma during the day, 3.1 +/- 0.2 pg/mL plasma versus 1.9 +/- 0.2 pg/mL plasma during the night). The 5-ML/melatonin ratio appeared much higher during the day than during the night but was very similar in both groups (day-time: 1.03 in the high group versus 1.16 in the low group, night-time: 0.01 in both groups). In a second experiment, six low group and seven high group ewes were submitted to 1 hr of extra light at night. 5-ML increased and melatonin decreased during extra light. Our results clearly show for the first time a daily variation in circulating 5-ML, and that the strong genetic contribution in the variability in melatonin concentrations in sheep are clearly correlated with a similar variability in 5-ML concentrations. Whether 5-ML, like melatonin, plays a physiological role in the different adaptation processes to the environment remains to be determined.

Increased sympathetic activity during sleep and nocturnal hypertension in Type 2 diabetic patients with diabetic nephropathy.

AIMS: To elucidate the putative factors involved in the blunted nocturnal blood pressure reduction in hypertensive Type 2 diabetic patients with diabetic nephropathy. METHODS: Extracellular fluid volume and fluid shift from interstitial to plasma volume (haematocrit), sympathetic nervous activity (plasma noradrenaline and adrenaline) and the internal 'body clock' (serum melatonin) were investigated in 31 hypertensive Type 2 diabetes mellitus (DM) patients with diabetic nephropathy (24 males, age 60 (45-73) years). All variables, except extracellular volume, were measured repeatedly with the patients lying awake in bed from 21:30 to 23:00 h (baseline) and during sleep from 23:00 to 07:00 h. Using the median nocturnal blood pressure reduction (8.4%) as a guide, the patients were divided into groups; group 1 with the highest and group 2 with the lowest nocturnal blood pressure reduction. RESULTS: Haematocrit decreased from baseline to the sleep period in group 1 by a mean (95% confidence interval (CI)) of 1.7 (0.3-3.1)%, but it increased by 0.5 (-1.0-1.9)% in group 2, mean difference (95% CI), -2.1 (-4.0 to -0.2)% (P = 0.029). Noradrenaline decreased from baseline to the sleep period, mean (95% CI), by 13.3 (0.0-25.0)% in group 1 but rose by 7.7 (-9.7-28.4)% in group 2, mean difference (95% CI), -19.6 (-35-0.0)% (P = 0.049). The nocturnal blood pressure change correlated to the nocturnal change in both noradrenaline (r = 0.51, P = 0.004) and haematocrit (r = 0.42, P = 0.018). Adrenaline remained constant in both groups. Extracellular fluid volume and plasma melatonin levels were comparable in the two groups. CONCLUSION: Sustained adrenergic activity during sleep is associated with blunted nocturnal blood pressure reduction in hypertensive Type 2DM patients with diabetic nephropathy, probably mediated through a lack of peripheral vasodilatation whereas changes in extracellular fluid volume distribution and melatonin secretion have no impact.

Immunohistochemical evidence for the presence of tryptophan hydroxylase and serotonin in the rodent Harderian gland.

The Harderian gland is considered as being an extrapineal source of melatonin. In most rodents, the Harderian gland contains two epithelial cell types (I and II). The aim of this study has been to define which cell type is involved in indoleamine synthesis. The presence and localization of serotonin (melatonin precursor) and tryptophan hydroxylase (the rate-limiting enzyme for serotonin synthesis) have been investigated by immunohistochemistry in male Wistar rats, Syrian hamsters and Djungarian hamsters. The results of the present study show that immunoreactivity for tryptophan hydroxylase and serotonin is confined to the type I cell, suggesting that this cell type is involved in indoleamine synthesis in the rodent Harderian gland.